缝隙连接蛋白43在受照人血管内皮细胞中的改变及其对受照细胞刚性的作用

目的 研究X射线对人脐静脉内皮细胞（HUVEC）中缝隙连接蛋白43（Cx43）的表达、分布和细胞刚性的影响,初步探讨Cx43对受照细胞刚性的调控作用。方法 采用Western blot方法检测10 Gy X射线照射后不同时间（0、6、12、24和48 h）和不同剂量X射线（0、2.5、5、10和20 Gy）照射后12 h HUVEC细胞中Cx43表达水平的改变,以及不同剂量（0、5和10 Gy） X射线照射后不同时间（3、6、24和48 h） Cx43 3个磷酸化位点（Ser279/282、Ser368和Tyr265）磷酸化水平的变化。采用细胞免疫荧光方法检测Cx43蛋白在受照HUVEC细胞中的分布变化。采用原子力显微镜检测受照细胞在探针压入不同深度（50、100和200 nm）时杨氏模量（细胞刚性）的变化,以及Cx43过表达对受照细胞杨氏模量（细胞刚性）的影响。结果 10 Gy X射线照射后6、12、24、48 h,HUVEC细胞中Cx43表达量降低（t=3.262、3.708、3.686、6.825,P<0.05）,且在2.5、5、10和20 Gy照射后24 h Cx43表达水平的降低与受照剂量呈现剂量依赖性（t=3.034、10.720、13.130、13.650,P<0.05）。5、10和20 Gy X射线照射后24 h,HUVEC细胞中Cx43分布由细胞间隙转移入核及核周,照射后24和48 h,Cx43的Ser368位点磷酸化水平升高并且随剂量增加而增加。10 Gy X射线照射HUVEC细胞后24 h,照射组与对照组相比,在探针压入100和200 nm时,杨氏模量均明显降低（t=3.362、5.122,P<0.05）;过表达Cx43的受照组与空载体受照组相比,在探针压入100和200 nm时,杨氏模量增高（t=2.674、4.398,P<0.05）。结论 X射线照射可导致HUVEC细胞内Cx43 Ser368位点磷酸化,促进Cx43降解和分布变化,降低细胞刚性。提高Cx43的表达水平,有助于受照细胞刚性的恢复,提示Cx43可能是调控X射线致血管内皮细胞损伤的作用靶点。     

In Vivo and In Vitro Expression of Connexin 43 in Human Teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of small regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.     

Drug development in dementia

Dementia is a progressive, irreversible decline in cognition that, by definition, impacts on a patient's pre-existing level of functioning. The clinical syndrome of dementia has several aetiologies of which Alzheimer's disease (AD) is the most common. Drug development in AD is based on evolving pathophysiological theory. Disease modifying approaches include the targeting of amyloid processing, aggregation of tau, insulin signalling, neuroinflammation and neurotransmitter dysfunction, with efforts thus far yielding abandoned hopes and ongoing promise. Reflecting its dominance on the pathophysiological stage the amyloid cascade is central to many of the emerging drug therapies. The long preclinical phase of the disease requires robust biomarker means of identifying those at risk if timely intervention is to be possible.     

Human Coronavirus OC43 Interacts with Major Histocompatibility Complex Class I Molecules at the Cell Surface to Establish Infection

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5x10⁶) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10⁸ PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9% Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes co-precipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.     

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20–25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the “nineteen complex” associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.     

A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely—not only in the nucleus, but also in the cytoplasm—and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.     

Spatio‐Temporal Development of Axonopathy in Canine Intervertebral Disc Disease as a Translational Large Animal Model for Nonexperimental Spinal Cord Injury

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of β‐amyloid precursor protein, non‐phosphorylated neurofilament (n‐NF) and growth‐associated protein‐43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n‐NF. Real‐time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)‐2 and MMP‐9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.     

Enhanced connexin 43 expression following neural stem cell transplantation in a rat model of traumatic brain injury

Introduction

Reestablishment of functional networks after traumatic brain injury (TBI) has been proffered as one of the goals of neural stem cell (NSC) transplantation therapeutics. Gap junctions provide essential means for direct cellular communication by transferring small molecules and ions, which may provide insights into the interplay between grafted NSCs and host cells.

Material and methods

Thirty-six adult male Wister rats were used in this study. The controlled cortical impact (CCI) model of brain injury has been performed. Seventy-two hours after CCI injury, animals were randomly assigned to two groups: PBS- and NSC- transplanted group. NSCs-transplanted group received delivery of the NSCs suspension to the cortex below the injury cavity in the ipsilateral hemisphere. At 1, 2, and 4 weeks post-transplantation, we investigated the expression patterns of gap junction-associated connexin 43 (Cx43) in the transplant site and the border of CCI by immunohistochemistry, Western blot and RT-PCR.

Results

Our findings showed that Cx43 staining was significantly greater in the transplant site and the border of CCI in the NSCs-transplanted rats compared to the control rats at different time points (p < 0.01 at 1 week, p < 0.05 at 2 and 4 weeks). Significantly higher gene and protein expression of Cx43 was found in NSCs-transplanted rats compared to the control rats in the period of 4 weeks post-transplantation (p < 0.01), and remained at a higher level until 2 weeks with or without NSC transplantation.

Conclusions

It is proposed that gap junction-associated Cx43 might participate in NSCs’ beneficial effects via gap-junctional coupling by which grafted NSCs integrate into host neural tissue following transplantation after TBI.     

Inhibition of GAP-43 by propentofylline in a rat model of neuropathic pain

Neural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. The relation of growth-associated protein-43 (GAP-43), a protein involved in the nerve fiber growth and sprouting, to pain hypersensitivity has been investigated. Glial activation and inflammatory cytokines released by microglia and astrocytes are considered to be involved in the neural sprouting and plasticity. In the present study, the anti-nociception effect of propentofylline, a glial modulating agent, was investigated in a rat chronic constriction injury (CCI) model aiming to explore the role of GAP-43 expression. Our results demonstrated that propentofylline could attenuate the CCI-induced mechanical allodynia and thermal hyperalgesia and inhibit the astrocyte activation and production of IL-1β. GAP-43 expression was also down-regulated by intrathecal propentofylline. These findings suggest that astrocyte activation is involved in the regulation of GAP-43 expression and propentofylline might be used in the treatment of neuropathic pain.     

Clinical usefulness of immunohistochemistry for plakoglobin,N-cadherin,and connexin-43 in the diagnosis of arrhythmogenic right ventricular cardiomyopathy

Diagnosing arrhythmogenic right ventricular cardiomyopathy (ARVC) is often challenging because no single diagnostic tool is available to detect the disease. We evaluated whether analysis of plakoglobin, N-cadherin, and connexin-43 immunoreactivity can be used as a significant test in diagnosis of ARVC. We selected subjects with suspicion of ARVC (n=22) in patients who underwent endomyocardial biopsy (EMB) in Kyungpook National University Hospital (n=1326). The patients (n=22) were classified into definite ARVC patients (n=17) and borderline ARVC (n=5). We selected control subjects (n=20) who were autopsied and died of non-cardiac disease. Hematoxylin-eosin, Masson’s trichrome, and immunohistochemical stains for plakoglobin, N-cadherin, and connexin-43 were used for all specimens. Reduced immunoreactivity of plakoglobin was observed in 13 (76%) of the 17 patients with a definite ARVC and in 4 (80%) of the 5 patients with a borderline ARVC. All subjects displayed no significant reduction of the immunoreactivity for connexin-43 as well as for N-cadherin. Our investigation revealed that the immunohistochemical analysis for plakoglobin had an accuracy of 81%, 76% sensitivity, and 84% specificity in diagnosis of ARVC. Results of our study showed that the immunohistochemical analysis of plakoglobin had a relatively high sensitivity and specificity in ARVC, but immunohistochemistry for plakoglobin alone could not be relied upon as a diagnostic test for ARVC. We confirmed that N-cadherin and connexin-43 had no diagnostic value in ARVC.